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Fig. S1. Autophagosomes fuse with M. marinum-containing compartment. Raw GFP-LC3 infected with M. marinum RFP for 1 h (uptake) followed by 2 h chase was analysed by live cell imaging. Images of the different time points are depicted in this figure. A phagosome harbouring M. marinum fuses with an autophagosome (AP, white arrow) in close proximity. Scale bars: 5 μm.

Fig. S2.M. marinum sheltered in GFP-LC3 structures lacks ubiquitin labelling.

A. Raw GFP-LC3 cells were infected with M. marinum as described in Fig. 1 and ubiquitin was detected by immunofluorescence using an appropriate antibody. Three situations were observed: (i) bacteria surrounded by GFP-LC3 with no ubiquitin labelling (a–e), (ii) bacteria surrounded by GFP-LC3 with a strong ubiquitin labelling of the pathogen (f–j), and (iii) bacteria surrounded by GFP-LC3 with some ubiquitin labelling detected at the limiting membrane of the GFP-LC3-decorated phagosome but absent inside that compartment (k–o).

B. Quantification of the frequency of the observed situations described in (A) is depicted in this panel.

Scale bars: 10 μm.

Fig. S3.M. marinum-containing phagosomes do not fuse with lysosomes. (A) Macrophage lysosomes were labelled with colloidal-gold particles coated with mannose-BSA (BSA-Au) which were previously incorporated by Raw cells (see Experimental procedures). Typical single-membrane phagosomes harbouring a few bacteria devoid of BSA-Au are shown. Lysosomes are easily recognized as they are loaded with gold particles (black arrowheads, A, B and C). Occasionally a few bacteria phagosomes that fused with lysosomes were observed (C and D).

Fig. S4. Phagosome labelling with Lysotracker markedly decreased at 8 h of chase.

A. Raw GFP-LC3 cells were infected with live Mm RFP for 1 h, and chased for 2 h or 8 h (A) in full medium (A, a–d), full medium in the presence of rapamycin (A, i–l), or full medium in the presence of rapamycin plus wortmannin (A, m–p). As a control we also infected Raw GFP-LC3 cells with HK Mm (A, e–h). Cells were labelled with Lysotracker as indicated in Experimental procedures. Scale bars: 10 μm.

B. Quantification showing the percentage of phagosomes labelled by Lysotracker at 2 (black bars) and 8 h (white bars). A clear decrease between 2 and 8 h is observed for live Mm, suggesting phagosomal membrane damage. No changes on Lysotracker colocalization between 2 and 8 h were observed for HK Mm. Likewise, in the presence of Rp no decrease on Lysotracker labelling between 2 and 8 h of chase was observed. In contrast, when the Rp effect was inhibited with Wm, we observe almost a 50% decrease on bacteria colocalization at 8 h, respect to the 2 h chase. Data are representative of two independent experiments.

C. Raw GFP-LC3 macrophages were infected with M. marinum for 1 h, and chased for additional 2 h under different conditions: full nutrient medium (Ctrl) (C, a–d), full nutrient medium with the addition of Rp (Ctrl + Rp) (C, e–h) and full medium with the addition of Rp plus Wm (Rp + Wm) (C, i–l).

D. Colocalization with LC3 (hatched bars), LC3 and Cathepsin D (black bars) and Cathepsin D (grey bars) was determined, and the quantification is shown in this panel. Data are the mean ± SEM of three independent experiments. Significantly different from the control: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S5.M. marinum (Mm) induces processing of endogenous LC3.

A. Western blot showing LC3 processing for Raw cells incubated for 1 h with full nutrient medium (Ctrl), starvation medium (Stv), or infected with M. marinum wt (Mm), HK-M. marinum wt (HK-Mm), M. bovis BCG or M. marinum ΔRD1, is depicted in this panel.

B. Quantification of the levels of LC3-II with respect to tubulin levels is shown in this panel. Data represent the mean ± SEM of two independent experiments.

C. Raw GFP-LC3 macrophages infected with M. bovis BCG for 1 h and chased for 2 h in full medium conditions. No interaction with LC3 is observed.

Scale bars: 10 μm.

Movie S1. Autophagosomes fuse with M. marinum-containing phagosomes. Raw GFP-LC3 macrophages infected with M. marinum RFP (Mm) for 1 h (uptake), followed by 2 h chase, were analysed by live cell imaging. An autophagosome (AP) is observed approaching, docking and finally fusing with the Mm-containing phagosome, leading to the formation of a typical structure decorated with LC3. First both channels (GFP and RFP) are shown in the movie. In the second part of the movie only the GFP channel is shown in order to have a better visualization of the fusion event and the formation of the LC3-decorated phagosome.

FilenameFormatSizeDescription
CMI_1581_sm_fS1.tif1195KSupporting info item
CMI_1581_sm_fS2.tif3612KSupporting info item
CMI_1581_sm_fS3.tif8985KSupporting info item
CMI_1581_sm_fS4.tif4340KSupporting info item
CMI_1581_sm_fS5.tif694KSupporting info item
CMI_1581_sm_mS1.mpg10866KSupporting info item

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